Within the biotechnology industry, the purification of proteins on a commercial scale is an important challenge to the development of recombinant proteins for therapeutic and diagnostic purposes. Problems related to yield, purity, and throughput plague the manufacturing sector. With the advent of recombinant protein technology, a protein of interest can be produced using cultured eukaryotic or prokaryotic host cell lines engineered to express a gene encoding the protein. What results from the host cell culturing process, however, is a mixture of the desired protein along with impurities that are either derived from the protein itself, such as protein variants, or from the host cell, such as host cell proteins. The use of the desired recombinant protein for pharmaceutical applications is contingent on being able to reliably recover adequate levels of the protein from these impurities.
Conventional protein purification methods are designed to separate the protein of interest from impurities based on differences in size, charge, solubility, and degree of hydrophobicity. Such methods include chromatographic methods such as affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, and hydroxyapatite chromatography. These methods often employ a separation medium that can be designed to selectively adhere either the protein of interest or the impurities. In the bind-elute mode, the desired protein selectively binds to the separation medium and is differentially eluted from the medium by different solvents. In the flow-through mode, the impurities specifically bind to the separation medium while the protein of interest does not, thus allowing the recovery of the desired protein in the “flow-through.”
Current methods for the purification of proteins, such as antibodies, include two or more chromatographic steps. For example, the first step in the protein purification protocol often involves an affinity chromatography step that utilizes a specific interaction between the protein of interest and an immobilized capture reagent. Protein A adsorbents are particularly useful for affinity capture of proteins, such as antibodies, which contain an Fc region. However, drawbacks to using Protein A chromatography for protein purification include leakage of the Protein A capture agent, leading to contamination of the eluted protein product. Additionally, affinity capture does not separate protein variants, such as aggregated forms of the protein, from the protein of interest.
Researchers have used bind-elute methods, flow-through methods, and displacement methods in efforts to recover proteins free from impurities resulting from both the culturing process and from possible prior steps in the purification process itself. Examples of groups using a bind-elute step as a typical second step to purifying proteins after an affinity capture step include: U.S. Pat. No. 4,983,722, describing a bind-elute ion exchange method of reducing Protein A from a mixture; U.S. Pat. No. 5,429,746, describing a bind-elute hydrophobic interaction chromatography method for purifying IgG antibody from a mixture including Protein A impurities; and U.S. Pat. No. 5,644,036, describing a three-step process for obtaining a purified IgG antibody preparation comprising a Protein A step, a bind-elute ion exchange step, and a size exclusion step. Other groups have used a flow-through step after the affinity chromatography step. For example, PCT publication WO 04/076485 describes a method for removing leaked Protein A from an antibody purified by a Protein A chromatography step followed by a flow-through ion exchange step. PCT publication WO 03/059935 describes a method for purifying a protein in a sample comprising subjecting the sample to a flow-through hydroxyapatite chromatography step following an affinity chromatography step.
Other groups have used a single polishing-step purification scheme to avoid the problems associated with prior purification steps. For instance, U.S. Pat. No. 6,177,548 describes a single-step flow-through ion exchange method for removing aggregates from a biological sample where the pH of the sample is adjusted to 0.2 logs below the isoelectric point of the biological sample. U.S. Pat. No. 5,451,662 describes a single-step bind-elute ion exchange method where the pH of the crude protein mixture is adjusted to a point between the ranges of isoelectric points of the protein fractions to be separated. PCT publication WO 05/044856 describes a single-step displacement method for removal of high molecular weight aggregates from antibody preparations using hydroxyapatite chromatography.
None of the conventional bind-elute or flow-through methods in the prior art, however, is able to meet the needs of the biotechnology industry in terms of all the requirements of throughput, yield, and product purity. Bind-elute methods and displacement methods are limited by, among other factors, the capacity limit of the separation medium for the desired protein. Flow-through methods, on the other hand, do allow for higher load challenges than bind-elute methods but are limited by the capacity of the separation medium for the impurities. With flow-through methods, no substantial binding of the product to the column occurs; any substantial product binding is seen as negatively impacting product recovery. There is still a need for methods of recovering purified proteins at high throughput that meet the requirements for purity and yield necessary for therapeutic and diagnostic applications. In addition, commercial manufacturing processes add the needs for reliable, robust, and cost-effective purification schemes.